WO1997019180A2 - Vecteur constitue d'une sequence d'adn de regulation de transcription liee a une sequence d'adn codant pour une beta-lactamase pour therapie a base d'un promedicament enzymatique - Google Patents

Vecteur constitue d'une sequence d'adn de regulation de transcription liee a une sequence d'adn codant pour une beta-lactamase pour therapie a base d'un promedicament enzymatique Download PDF

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WO1997019180A2
WO1997019180A2 PCT/GB1996/002845 GB9602845W WO9719180A2 WO 1997019180 A2 WO1997019180 A2 WO 1997019180A2 GB 9602845 W GB9602845 W GB 9602845W WO 9719180 A2 WO9719180 A2 WO 9719180A2
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molecular
lactamase
sequence
targetted
chimeara
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PCT/GB1996/002845
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WO1997019180A3 (fr
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Inderjit Dev
John Tomlin Moore
Carol-Ann Dinsmore Ohmstede
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Glaxo Group Limited
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Priority to AU75839/96A priority Critical patent/AU7583996A/en
Publication of WO1997019180A2 publication Critical patent/WO1997019180A2/fr
Publication of WO1997019180A3 publication Critical patent/WO1997019180A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
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    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • C—CHEMISTRY; METALLURGY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • A—HUMAN NECESSITIES
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    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to targetted enzyme prodrug therapy.
  • Targetted enzyme prodrug therapies provide a method for restricting the activity of a chemotherapeutic agent to a particular target site This is desirable when the systemic presence of the chemotherapeutic agent produce unwanted side effects
  • the technique is particularly applicable to the treatment of cancer where therapeutic regimes have previously involved the systemic introduction of highly cytotoxic compounds which exert their effect in a non-selective manner on both healthy and tumourogenic cells.
  • antrtumour agents which have differing degrees of efficacy.
  • Standard clinically useful agents include adriamycin, actinomycin D, methotrexate, 5-fluorouracil, cis-platinum, vincristine and vinblastine.
  • these presently available antrtumour agents are known to have various disadvantages such as toxicity to healthy cells and resistance of certain tumour types.
  • Other forms of therapy such as surgery, are known.
  • novel approaches and entities for cancer therapies are required if significant progress in the clinical management of this disease is to be achieved
  • Targetted enzyme prodrug therapies may offer significant improvements in cancer therapy, either alone or in combination with existing treatment regimes.
  • One such therapy relates to the use of molecular chimaeras, which encode a heterologous enzyme and, which are delivered to targetted cells. Intracellular expression of the enzyme allows catalysis of a subsequently administered prodrug to its active cytotoxic or cytostatic form.
  • the therapy is known as gene or virus directed enzyme prodrug therapy (GDEPT or VDEPT).
  • WO-A-90 07936 describes a treatment for an infection or a hyperprolrferative disorder which is characterised by the presence, in the affected cells, of a trans-acting factor capable of regulating gene expression by inserting into the cells a polynucleotide construct having a cis-acting regulatory sequence which is regulated by the transacting factor and an effector gene which renders said cell susceptible to protection or destruction
  • the cis-acting region may be homologous to the HIV tar region and the effector gene may encode ricin A or HSV-1 thymidine kinase
  • the HIV tat protein activates the tar region, and induces transcription and expression of nan A, resulting in cell death, or of HSV-1 tk, resulting in cell death upon treatment with dideoxynucleoside agents such as acyclovir and gancyclovir.
  • EP-A-0 334 301 describes methods for the delivery of vectors using recombinant retrovirus wherein the vector construct directs the expression of a protein that activates a compound with little or no cytotoxicrty into a toxic product in the presence of a pathogenic agent, thereby effecting localised therapy to the pathogenic agent.
  • EP-A-0 415 731 describes molecular chimaeras for use with prodrugs, comprising transcriptional regulatory DNA sequences capable of being selectively activated in a mammalian cell, a DNA sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a heterologous enzyme capable of catalysing the conversion of the prodrug into an agent toxic to the cell.
  • EP-0 382 411 proposes a targetted enzyme prodrug therapy for neoplastic disease which utilises antibodies to direct an enzyme such as ⁇ -lactamase to neoplastic tissues where the enzyme can catalyse conversion of a prodrug to a cytotoxic agent.
  • an enzyme such as ⁇ -lactamase
  • neoplastic tissues where the enzyme can catalyse conversion of a prodrug to a cytotoxic agent.
  • this approach is limited by the availability of suitable targetting antibodies and their lack of ability to penetrate solid tumours and by the fact that the cytotoxic agent is generated extracellularly and therefore must enter the neoplastic cell to exert its cytotoxic effect.
  • prodrugs suitable for use in GDEPT and hence the range of cytotoxic or other threapeutic agents which can be targetted using this approach, is limited by the availability of DNA encoding an enzyme which possesses appropriate catalytic activity to convert the prodrug into the active cytotoxic or other therapeutic agent, which DNA is capable of expression in a eukaryote .
  • An object of the present invention is to improve and extend the range of prodrugs which can be used in GDEPT.
  • the present invention relates to the use of a ⁇ -lactamase enzyme in GDEPT. More particularly the present invention provides a molecular chimaera for use in therapy with a prodrug, the chimeara comprising a transcriptional regulatory DNA sequence capable of being activated in a targetted mammalian call and a DNA coding sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a ⁇ - lactamase enzyme such that on expression of said coding sequence in the targetted cell, the ⁇ -lactamase enzyme is capable of catalysing conversion of the prodrug into an agent toxic to the targetted cell.
  • the enzyme ⁇ -lactamase has particular advantages for use in GDEPT in terms of the range of toxic agents which can be presented in the form of prodrugs capable of conversion to the active agent by means of the enzyme.
  • any toxic agent can be converted to such a prodrug by conjugation with another compound through a bond capable of being cleaved by ⁇ -lactamase.
  • conjugates are formed between the toxic agent and a cephalosporin.
  • toxic agents include 5-fluorouracil, methotrexate and adriamycin which may be linked in each case to, for example, a cephalosporin (see WO-A-94 01 137 and EP-A-0 382 411) or cephalosporin mustards (see EP-A-0 484 870).
  • cephalosporin/toxic agent conjugate shows markedly reduced toxicity but can be converted to the active form by ⁇ -lactamase thus making it suitable for use as a prodrug in GDEPT.
  • Other toxic agents can be linked to cephalosporins in a similar way.
  • Prodrugs for use according to the present invention may thus be based on any compound showing a suitable chemotherapeutic effect.
  • chemotherapeutic agents are preferably anti-inflammatory, anti-viral or anti-cancer compounds, and more preferably cytotoxic compounds such as nitrogen mustard agents, antifolates, nucleoside analogs, the vinca alkaloids, the anthracyclines, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, the podophyophyllotoxins, the sulfonylureas (as described in EP-A-0 222,475) and low-molecular-weight toxins such as the trci hothecenes and the colchicines.
  • cytotoxic compounds such as nitrogen mustard agents, antifolates, nucleoside analogs, the vinca alkaloids, the anthracyclines, the mitomycins, the bleomycins, the cytotoxic nucleo
  • doxorubicin particularly including doxorubicin, daunorubicin, aminopterin, methotrexate, taxol, methopterin, dichloromethotrexate, mitomycin C, porfirmoycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, melphalan, vinblastine, vincristine, desacetylvinblastine hydrazide, leurosidine, vindesine, leurosine, trichothecene and desacetylcolchicine.
  • the molcular chimeara according to the present invention may provide intracellular expression or membrane bound expression or secretion of the ⁇ -lactamase enzyme in the targetted cell.
  • Secretion or membrane bound expression of the ⁇ -lactamase enzyme has the advantage of increasing the phenomenon known as "neighbouring cell kill”.
  • GDEPT and VDEPT regimes which involve the intracellular expression of the heterologous enzyme and intracellular catalysis of the prodrug are limited in terms of their efficiency in that, for example in tumour therapy using retroviral mediated targetting, only between 1 and 10% of cells of the solid tumour may be infected by retrovirus. Hence therapy is limited to those 1 to 10% of infected cells and the neighbouring 90-99% of cells remain untreated.
  • an enzyme should possess a signal sequence at the amino terminus either because it is a secreted enzyme with a naturally occurring signal sequence or because the chimaera expressing the enzyme has been engineered such that the expressed enzyme has an additional amino acid sequence which possesses the properties of a signal sequence.
  • the molecular chimaera should also include at a suitable position DNA sequence encoding a membrane anchoring peptide.
  • peptides may be dervi ed, for example, from proteins with C-terminal domains substituted with phospholipid anchors.
  • proteins include Thy-1 (Low et al, Nature, 318, 62 (1985) and Tse et al, Science, 230, 1003 (1985)), the variant surface glycoproteins (VSGs) of African trypanosomes (Ferguson et al, J. Biol.
  • the coding sequence is under the control of a transcriptionai regulatory sequence (TRS) comprising at least a promoter and preferably an enhancer, each of which may either be capable of non-specific expression independent of the type of cell in which expression is occurring or may exhibit a selectivity of expression dependent upon the cellular environment
  • TRSs are non-specific, potent promoter/enhancer combinations such as cytomegalovirus promoter/enhancer, SV40 promoter/enhancer and retroviral long terminal repeat promoter/enhancer
  • Other preferred TRSs include those of ⁇ -actin, glyceraldehyde-S-phosphate and tubulin.
  • TRSs exhibiting cell-type dependent, for example, tissue specific or tumor specific, expression in which case the selection of the TRS, in particular the promoter and enhancer sequence, will depend on the targetted cell type
  • examples include the albumin (ALB) and alpha-fetoprotein (AFP) TRS for normal hepatocytes and transformed hepatocytes respectively, the TRS for carcinoembryonic antigen (CEA) for use in transformed cells of the gastrointestinal tract, lung, breast and other tissues: the TRS for tyrosine hydroxylase, cholme acetyl transferase or neuron specific enolase for use in neuroblastomas the TRS for glial fibro acidic protein for use in glioblastomas and the TRS for insulin for use in tumours of the pancreas.
  • ALB albumin
  • AFP alpha-fetoprotein
  • CEA carcinoembryonic antigen
  • TRS specific for gamma-glutamyltranspeptidase for use in certain liver tumours and dopa decarboxylase for use in certain tumours of the lung
  • the present invention is also useful in treatment of ovarian cancer, colon cancer, breast cancer, prostrate cancer and melanomas.
  • TRS for certain oncogenes may be used as these are expressed predominantly in certain tumour types. These include the HER-2/neu oncogene TRS which is expressed in breast tumours and the TRS specific for the N-myc oncogene for neuroblastomas.
  • the ALB and AFP genes exhibit extensive homology with regard to nucleic acid sequence, gene structure, amino acid sequence and protein secondary folding (for review see Ingram et al Proc. Natl. Acad. Sci. (USA) 78, 4694-4698 (1981)). These genes are independently but reciprocally expressed in ontogeny. In normal development ALB transcription is initiated shortly before birth and continues throughout adulthood. Transcriptional expression of ALB in the adult is confined to the liver.
  • AFP is normally expressed in foetal liver, the visceral endoderm of the yolk sac and the foetal gastrointestinal tract, but declines to undetectable levels shortly after birth and is not significantly expressed in nonpathogenic or non-regenerating adult liver or in other normal adult tissues.
  • AFP transcription in adult liver often increases dramatically in hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • transcription may also be elevated in non-seminomatous and fixed carcinoma of the testis: in endodermal sinus tumours in certain tertorcarcinomas and in certain gastrointestinal tumours.
  • Liver-specific expression of AFP and ALB is the result of interactions of the regulatory sequences of their genes with trans-activating transcriptional factors found in nuclear extracts from liver.
  • AFP and ALB TRSs are preferred for generating hepatoma-specific or general liver-specific expression respectively of moleculariy combined genes since the AFP and ALB genes are regulated at the transcriptional level and their mRNAs are among the most abundant polymerase II transcripts in the liver.
  • the regulatory elements of the AFP genes promote tissue-specific expression in certain liver pathologies, such as HCC (Mol. Cel. Biol. 6, 477-487 (1986): Science, 235, 53-58 (1987)).
  • the regulatory elements of a mammalian AFP gene consist of a specific 5' promoter proximal region (located in some mammalian species between 85 and 52 bp 5' to the gene). This sequence is essential for transcription in hepatomas.
  • upstream (5') regulatory elements well defined for the murine AFP gene which behave as classical enhancers (Mol. Cel. Biol. 6, 477-487 (1986): Science, 235, 53-58 (1987)).
  • upstream regulatory elements are designated elements I, II and III and are located between 1,000 to 7,600 bp 5' to the transcription initiation site for the AFP murine gene. These three enhancer domains are not functionally equivalent at generating tissue-specific expression of AFP. Elements I and II have the greatest capacity to direct liver-specific expression of AFP. It is important to note that the regulatory sequences of the alpha-fetoprotein gene advantageously contain the sequences not only for tissue-specific transcriptional activation but also for repression of expression in tissues which should not express AFP. In a similar fashion the regulatory regions of the human alpha-fetoprotein gene have been characterised (J. Biol. Chem. 262, 4812-4818 (1987)).
  • a structural gene placed in the correct orientation 3' to the AFP regulatory sequences will enable that structural gene to be selectively expressed in fetal liver hepatomas, non-seminamatous carcinomas of the testis, certain teratocarcinomas, certain gastrointestinal tumours and other normal and pathological tissues which specifically express AFP.
  • the promoter and enhancer sequences preferably are selected from the TRS for one of albumin (ALB), alphafetoprotein (AFP), carcinoembryonic antigen (CEA) (J. DNA Sequencing and Mapping, Vol 4, 185-196), tyrosine hydroxylase, choline acetyl transferase, neuron-specific enclase, glial fibro acid protein, insulin or gamaglutamytranspeptidase, dopadecarboxylase, HER-2/neu or N-myc oncogene or other suitable genes.
  • ALB albumin
  • AFP alphafetoprotein
  • CEA carcinoembryonic antigen
  • TRS for ALB or AFP are used to direct liver specific or hepatoma specific expression respectively.
  • the molecular chimaera is selectively expressed in a target cell population. This may be taken to mean that the chimaera is expressed at a higher level in the target than in the non-target cell population and is preferably expressed predominantly or exclusively in that population.
  • Selective expression may be achieved by inclusion of a target-cell specific TRS
  • promoter with or without enhancer may be a product of the method of delivery of the chimaera to the target cell.
  • Methods capable of providing target cell specific delivery of the chimaera, with subsequent stable integration and expression include the techniques of calcium phosphate transfection, electroporation, microinjection, liposomal transfer, ballistic barrage or retroviral infection or infection using adenovirus or adeno-associated virus.
  • selectivity may be obtained by a vanety of such techniques .
  • Physiologically localised delivery of the chimaera for the target cells will reduce the possibility of non-target cells expressing the chimaera.
  • Retroviral or liposome mediated delivery may involve direct injection to a blood vessel known to supply the target cells.
  • Selectivity may also be obtained using retroviral mediated chimaera delivery in the therapy of hyperprolrferative disorders. Retroviruses only infect dividing cells and would therefore only introduce chimaeras to dividing cells.
  • Liposome technology permits the delivery of the chimaera contained therein to be targetted to a particular cell type based on appropnate modifications made to the liposome coat structure. In a preferred embodiment according to the present invention, a number of such methods for obtaining selectivity will be combined to improve the fidelity of selective expression.
  • the chimaera may comprise TRSs derived from Iiver-specific gene promoters such as ALB or AFP, and will be delivered in a retrovirus directly to the hepatc artery.
  • TRSs derived from Iiver-specific gene promoters such as ALB or AFP
  • a retrovirus which only infects dividing carcinoma cells
  • liver-specific expression of the TRSs One particular method according to the present invention for obtaining selective expression of a molecular chimaera of the present invention delivered using a retrovirus is accomplished by promoting selective infection of liver cells.
  • This technique involves the retroviral env gene present in the packaging cell line which defines the specificity for host infection.
  • the env gene used in constructing the packaging cell line is modified to generate artificial infective virions that selectively infect hepatocytes.
  • a retroviral env gene introduced into the packaging cell may be modified in such a way that the artificial infective virion's envelope glycoprotein selectively infect hepatocytes via the specific receptor mediated binding utilised by the hepatitis B virus (HBV) HBV pnmanly infects hepatocytes via specific receptor mediated binding
  • HBV proteins encoded by the pre-S1 and pre-S2 sequences play a major role in the attachment of HBV to hepatocytes (see Hepadna Viruses edited Robinson et al 189-203, 205-221 (1987)).
  • the env gene of the packaging cell is modified to include the hepatocyte binding site of the large S HBV envelope protein
  • modifications of the env gene introduced into the packaging cell may be perfomned by standard molecular biology techniques well known in the art and will facilitate viral uptake in the target tissue.
  • the TRS need not be target cell specific and TRSs dervi ed from genes such as ⁇ -actin, glyceraldehyde-3-phosphate and cytomegalovirus (e.g. immediate early gene) (see Huber, et al Cancer Research, 53, 4619-4626 (1993) and references therein) may be used.
  • genes such as ⁇ -actin, glyceraldehyde-3-phosphate and cytomegalovirus (e.g. immediate early gene) (see Huber, et al Cancer Research, 53, 4619-4626 (1993) and references therein) may be used.
  • the molecular chimaera of the present invention may be made utilising standard recombinant DNA techniques.
  • the coding sequence and polyadenylation signal of for example the cytosine deaminase gene is placed in the proper 3' orientation to the ALB or AFP TRS.
  • These molecular chimaeras enable the selective expression of cytosine deaminase in cells which normally express from ALB or AFP TRSs respectively.
  • molecular chimeara have been constructed for secretion, intracellular expression and transmembrane expression of ⁇ -lactamase in a mammalian cell.
  • DNA sequences from/these constructs are set out in SEQ IDs Nos 3,
  • a method of constructing a molecular chimaera comprising operatively linking a DNA sequence comprising a TRS capable of being activated in a mammalian cell to a DNA sequence encoding a ⁇ -lactamse enzyme capable of expression in a mammalian cell.
  • the present invention provides a molecular chimeara as defined above wherein the sequence providing for intracellular expression of the ⁇ -lactamase enzyme is at least 80% homologous to that of SEQ ID NO 5.
  • the sequence providing for intracellular expression of the ⁇ -lactamase enzyme has the sequence of SEQ ID NO 5.
  • the present invention provides a molecular chimeara as defined above wherein the sequence providing for secretion of the ⁇ -lactamase enzyme is at least 80% homologous to that of SEQ ID NO 3.
  • the sequence providing for secretion of the ⁇ -lactamase enzyme has the sequence of SEQ ID NO 3.
  • the present invention provides a molecular chimeara as defined above wherein the sequence providing for membrane bound expression of the ⁇ -lactamase enzyme ia at least 80% homologous to that of SEQ ID NO 9.
  • the sequence providing for membrane bound expression of the ⁇ -lactamase enzyme has the sequence of SEQ ID NO 9.
  • retroviral shuttle vectors which are known in the art (see for example Mol. and Cell Biol. 6, 2895-2902 (1986)).
  • retroviral shuttle vectors are generated using the DNA form of the retrovirus contained in a plasmid. These plasmids also contain sequences necessary for selection and growth in bacteria.
  • Retroviral shuttle vectors are constructed using standard molecular biology techniques well known in the art. Retroviral shuttle vectors have the parental endogenous retroviral genes (e.g. gag, pol and env) removed and the DNA sequence of interest inserted, such as the molecular chimaeras which have been described.
  • Retroviral shuttle vectors have been derived from the Moloney murine leukaemia virus (Mo-MLV) but it will be appreciated that other retroviruses can be used such as the closely related Moloney murine sarcoma virus.
  • Mo-MLV Moloney murine leukaemia virus
  • retroviruses can be used such as the closely related Moloney murine sarcoma virus.
  • Certain DNA viruses may also prove to be useful as a delivery system.
  • the bovine papilloma virus (BPV) replicates extrachromosomally so that delivery system based on BPV have the advantage that the delivered gene is maintained in a nonintegrated manner.
  • Adenoviruses and adeno-associated viruses may also be used.
  • the advantages of a retroviral-mediated gene transfer system are the high efficiency of the gene delivery to the targeted tissue sequence specific integration regarding the viral genome (at the 5' and 3' long terminal repeat (LTR) sequences) and little rearrangements of delivered DNA compared to other DNA delivery systems.
  • LTR long terminal repeat
  • a retroviral shuttle vector comprising a DNA sequence comprising a 5' viral LTR sequence, a cis acting psi encapsidation sequence, a molecular chimaera as hereinbefore defined and a 3' viral LTR sequence.
  • the molecular chimaera is placed in opposite transcriptional orientation to the 5' retroviral LTR.
  • a dominant selectable marker gene may also be included which is transcriptionally drvi en from the 5' LTR sequence.
  • Such a dominant selectable marker gene may be the bacterial neomycin-resistance gene NEO (aminoglycoside-3-phosphotransferase type II) which confers on eukaryotic cells resistance to the neomycin analogue G418 sulphate (Geneticin - trade mark).
  • NEO aminoglycoside-3-phosphotransferase type II
  • the NEO gene aids in the selection of packaging cells which contain these sequences.
  • the retroviral vector used may be based on the Moloney murine leukaemia virus but it will be appreciated that other vectors may be used. Such vectors containing a NEO gene as a selectable marker have been described, for example, the N2 vector (Science, 230, 1395-1398 (1985)).
  • retroviral shuttle vectors A theoretical problem associated with retroviral shuttle vectors is the potential of retroviral long terminal repeat (LTR) regulatory sequences transcriptionally activating a cellular oncogene at the site of integration in the host genome. This problem may be diminished by creating SIN vectors. SIN vectors are self-inactivating vectors which contain a deletion comprising the promoter and enhancer regions in the retroviral LTR.
  • LTR long terminal repeat
  • the LTR sequences of SIN vectors do not transcriptionally activate 5 or 3 genomic sequences.
  • the transcriptional inactivation of the viral LTR sequences diminishes insertionai activation of adjacent target cell DNA sequences and also aids in the selected expression of the delivered molecular chimaera SIN vectors are created by removal of approximately 299 bp in the 3 viral LTR sequence (Biotechniques, 4, 504-512 (1986)).
  • the retroviral shuttle vector of the present invention are SIN vectors.
  • helper virus system may be utilised to provide the gag pol and env retroviral gene products trans to package or encapsidate the retroviral vector into an infective virion. This is accomplished by utilising specialised "packaging" cell lines which are capable of generating infectious synthetic virus yet are deficient in the ability to produce any detectable wild-type virus. In this way the artificial synthetic virus contains a chimaera of the present invention packaged into synthetic artificial infectious virions free of wild-type helper virus.
  • helper virus that is stably integrated into the packaging cell contains the viral structural genes but is lacking the psi site and cis acting regulatory sequence which must be contained in the viral genomic RNA molecule for it to be encapsidated into an infectious viral particle.
  • the present invention provides an infective virion comprising a retroviral shuttle vector as hereinbefore described said vector being encapsidated within viral proteins to create an artificial infective replication-defective retrovirus.
  • helper virus LTR regulatory sequences In addition to removal of the psi site additional alterations can be made to the helper virus LTR regulatory sequences to ensure that the helper virus is not packaged in virions and is blocked at the level of reverse transcription and viral integration.
  • helper virus structural genes i.e. gag pol and env
  • helper virus structural genes may be individually and independently transferred into the packaging cell line. Since these viral structural genes are separated within the genome of the packaging cell, there is little chance of covert recombinations generating wild-type virus.
  • infective virions of the present invention by dehvering the artificial retroviral shuttle vector comprising a molecular chimaera of the invention as hereinbefore described into a packaging cell line.
  • the packaging cell line may have stably integrated within it a helper virus lacking a psi site and other regulatory sequence as hereinbefore described or alternatively the packaging cell line may be engineered so as to contain helper virus structural genes within its genome.
  • the present invention further provides an infective virion as hereinbefore described for use in therapy particularly for use in the treatment of cancer and more particularly for use in the treatment of hepatocellular carcinoma, non-seminamatous carcinoma of the testis, certain teratocarcmomas and certain gastrointestinal tumours.
  • the infective virion according to the invention may be formulated by techniques well known in the art and may be presented as a formulation with a pharmaceutically acceptable earner therefor.
  • Pharmaceutical acceptable carriers in this instance may comprise a liquid medium suitable for use as vehicles to introduce the infective virion into the patient.
  • An example of such a earner is saline.
  • the infective virion may be a solution or suspension in such a vehicle.
  • Stabilisers and antioxidants and or other excipients may also be present in such pharmaceutical formulations which may be administered to a mammal by any conventional method e.g. oral or parenteral routes.
  • the infective virion may be administered by intra-venous or intra-arterial infusion.
  • intra-hepatic arterial infusion may be advantageous.
  • the invention also provides pharmaceutical formulations comprising a molecular chimaera of the present invention contained within one of, an infective virion or a liposome or a packaging cell mix, in admixture with a pharmaceutically acceptable carrier, and pharmaceutical formulations comprising a molecular chimaera virion, vector, liposome or packaging cell mix of the present invention in admixture with a pharmaceutically acceptable carrier.
  • the present invention provides methods of making pharmaceutical formulations as herein described comprsi ing mixing an artificial infective virion containing a molecular chimaera with a pharmaceutically acceptable earner.
  • the invention also includes the use of any molecular chimaera, vector, virion, liposome or pharmaceutical formulation of the present invention in human therapy and in the manufacture of a medicament for use in the treatment of pathological states.
  • the invention also includes methods of medical therapy comprising the use of any molecular chimaera, vector, virion, liposome or pharmaceutical formulation of the present invention.
  • a protein encoded by a molecular chimaera of the present invention is also included within the scope of the present invention and any combination of such a protein and a prodrug which can be catalysed by the enzyme component of that protein.
  • the precise dosage to be administered to a patient will ultimately be dependent upon the discretion and professional judgement of the attendant physician and will be a product of the particular targetting mechanism chosen. References contained herein to the efficiency of targetting of retroviruses, liposome etc. may be used to determine appropriate dosage levels.
  • colony forming units per ml (CFU/ml) infective viroi ns is likely to be suitable for a typical tumour.
  • Total amount of virions infused will be dependent on tumor size and would probably be given in divided doses.
  • the dose of prodrug will advantageously be in the range of 0.1 to 250mg per kilogram body weight of recipient per day, preferably 0.1 to 100 mg per kilogram bodyweight.
  • the present invention further provides a method of treating cancer, a viral infection or an inflammatory condition which comprises administering an effective amount of chimeara, vector, packaging cell line or infective virion according to the invention.
  • the viral infection includes, for example, HIV, HBC, HCV and herpes family virus.
  • Figure 1 shows cellular location of ⁇ -lactamase activity in mammalian cells transfected with ⁇ -lactamse constructs
  • prodrugs of methotrexate (5798W93) and 5-fluorouracil (1614W94) have been synthesized, ⁇ -lactamase constructs have been created which give rise to secreted, intracellular and membrane-anchored forms.
  • the forward primer contains a Hind 111 restriction site (AAGCTT) for subsequent cloning of the PCR product, and a sequence (GCCACC) which confers optimal translation effciency in vertebrates (Kozak, J. Cell Biol. 115, 887-903 (1991)) immediately 5-prime to the initiator methionine codon (ATG) of the ⁇ -lactamase coding region.
  • the reverse primer contains an Xba I restriction site (TCTAGA) adjacent to the stop codon (TAA) of the ⁇ -lactamase coding region.
  • PCR reaction was carried out for 25 cycles using standard conditions and using Vent DNA Polymerase (New England Biolabs, Inc., Beverly, MA, USA) in 4 mM MgSO 4 and 200 ⁇ M of each dNTP and 1 pmol/ ⁇ l forward and reverse primers.
  • PCR thermal cycling conditions were 95°C, 1 min; 60°C, 1 min; 75°C, 1 min, 25 cycles then 75°C, 5 min.
  • the approximately 800 base pair PCR product was gel-purfiied using the Glass-Max kit (Life Technologies, Inc., Gaithersburg, MD, USA) .
  • the purified PCR product was restriction digested with Hind III and Xba I, re-punfied by gel electrophoresis, and ligated into the multiple cloning site of the pRc/CMV vector (InVitrogen, Inc., San Diego, CA).
  • the orientation of the ⁇ -lactamase insert in this vector places the ⁇ -lactamase gene under the transcriptional regulation of the intermediate/early CMV promoter as well as followed a bovine growth hormone poly(A) addition signal.
  • the sequence of the construct (designated pCMV-BL) is shown in SEQ ID NO 3 along with the ammo acid sequence of inserted secretory ⁇ -lactamase.
  • This forward primer consists of a Hind III restriction site (AAGCTT), a concensus site for optimal traslation efficiency (GCCACC) in vertebrates (Kozak, 1991 supra) and an ATG initiator codon immediately adjacent to the sequence representing the mature ammo-terminus of TEM ⁇ -lactamase (Sutclrffe, 1978 supra)
  • AAGCTT Hind III restriction site
  • GCCACC concensus site for optimal traslation efficiency
  • the resulting PCR product would contain a deleted signal peptide and a new initiator methionine codon adjacent to the mature coding region of ⁇ -lactamase.
  • This PCR reaction was earned out using PCR conditions identical to those described for pCMV-BL, except that JM30 was substituted for JM1
  • the approximately 700 base pair PCR product was gel-purified using the Glass-Max kit (Life Technologies, Inc., Gaithersburg, MD, USA).
  • the purified PCR product was restriction digested with Hind III and Xba I, repurified by gel electrophoresis, and ligated into the multiple cloning site of the pRc/CMV vector (InVitrogen, Inc., San Diego, CA, USA) as described above for pCMV-BL.
  • pCMV- ⁇ BL (designated pCMV- ⁇ BL) is shown in SEQ ID NO 5 along with the ammo acid sequence of inserted intracellular ⁇ -lactamase.
  • a membrane-spanning domain was appended to the carboxy-terminus of the secretory ⁇ -lactamase coding region contained in pCMV-BL.
  • the membrane sequence was derived from the human C mu IgM heavy chain gene (Dorai, Nucl. Acids Res., 17, 6412 (1989)). This was done by fusing a 300 base pair sequence representing the human IgM membrane-spanning domain (from plasmid IgM/TM/PCRII which contains exons M1 and M2 separated by a single intervening sequence) in-frame to the carboxy-terminus of the secretory ⁇ -lactamase gene.
  • the first step in this process was to delete the termination codon in the ⁇ -lactamase sequence contained in pCMV-BL. This was done by PCR amplification of the insert using the forward primer JM1 (see above) in combination with the reverse primer MEM1.
  • M EM 1 consists of the sequence
  • MEM1 contains sequence representing the carboxy-terminus of secretory ⁇ -lactamase excepting the translation termination signal (TAA) which is replaced by an Xba I restriction site.
  • TAA translation termination signal
  • Xba I restriction site The hexameric Xba I sequence is in-frame with the coding region of ⁇ -lactamase and represents a Ser-Arg amino acid sequence.
  • This PCR product was amplified as described above, gel-purified, and cloned into the Hind III and Xba I sites of pRc-CMV. This plasmid was designated pCMV-MEM1.
  • pCMV-MEM1 To attach a carboxy-terminal membrane spanning domain, a 300 base-pair sequence from plgM/TM/PCRll was amplified with oligos MEM2 and MEM3.
  • MEM2 consists of the sequence
  • MEM3 consists of the sequence 5'-TGACAAGGGCCCCTCTGGTCTCCGATGTTCTTC (33-mer) (SEQ ID NO 8).
  • MEM2 represents the ammo-terminus of the IgM trans-membrane domain (beginning at nucleotide 489; GenBank Accession #X14939) flanked by an Xba i restriction site (TCTAGA).
  • MEM3 represents the carboxy-terminus of the trans-membrane domain (ending at nucleotide 815; GenBank Accession #X14939) flanked by an Apa I restriction site (GGGCCC).
  • oligos were used to carry out PCR as described above and the approximately 300 base-pair product was restriction digested, gel-purified, and cloned into the Xba I and Apa I sites of pCMV-MEM1.
  • the sequence of the construct (designated pCMV-BLIgM) along with the amino acid sequence of inserted membrane-anchored ⁇ -lactamase is shown in SEQ ID NO 9.
  • transfected cells were resuspended in 50 mM Tris-CI (pH 7.4), 0.1 mM EDTA containing PMSF and leupeptin, swollen on ice for 10 mm, then lysed using a Dounce homogenizer. After centrifugation at 800 ⁇ g for 6 min, supernatant (cytosolic fraction) was recentrifuged at 30 psi for 20 minutes in a Beckman AirFuge. Pellets from both centrfiugations (which include membranes and nuclei) were combined.
  • a 500 ⁇ M PADAC stock was made in water, filtered through a 0.22 ⁇ m filter, and added to media to give a final concentration of 20 ⁇ M Decreases in absorbance at 570 nm were measured using the auto-rate assay of a Kontron UV/Vis spectrophotometer.
  • stable lines were generated for use in immunohistochemistry experiments.
  • large-scale transfections in A549 cells were performed. Since pCMV-BL, pCMV- ⁇ BL, and pCMV-BLIgM contain the neomycin R gene, stable lines could be selected after passaging the lines in media containing the antibiotic, G418.
  • Clonal lines were derived which secrete ⁇ -lactamase (pCMV-BUA549), lines which synthesize an intracellular ⁇ -lactamase (pCMV- ⁇ BUA549), and lines which synthesize membrane- bound ⁇ -lactamase (pCMV-BLIgM/A549).
  • Prodrugs of methotrexate (5798W93) and 5-fluorouracil (1614W94) represent the parent drugs linked to cephalothin.
  • the kinetic parameters of prodrug activation were measured by incubating various concentrations of prodrug with purfiied ⁇ -lactamase followed by HPLC analysis to determine the rate of prodrug conversion.
  • ⁇ -Lactamase efficiently activates both 5798W93 and 1614W94 with a k cat /K M , (specificity constant) of 272 and 67 sec -1 mM -1 , respectively.
  • methotrexate was 10-fold more toxic than the methotrexate prodrug 5798W93, and fluorouracil was 20-fold more toxic than the fluorouracil prodrug 1614W94 (Table 1).
  • A549 cells which contained stable integrated copie(s) of the secretory ⁇ -lactamase gene (A549-BL) were tested, methotrexate and its prodrug 5798W93 were equally toxic (Table 1). This experiment implies that the delivery of the ⁇ -lactamase gene to tumor cells will make them sensitive to cephalosporin prodrugs.
  • Prodrug therapy (1614W94 (50 mg/kg; i.p., qd x 5) or 5-FC (500 mg/kg; i.p., qd x 5) was initiated two days after DNA treatment. Inhibition of tumour growth was determined on day 47. Both CD and BL constructs resulted in similar antitumour activity in vivo. 1614W94 administration resulted in about 60% inhibition of tumour growth (Table 3). 5-FC administration resulted in about 70% inhibition of tumour growth, whereas DNA liposomes alone and 5-FU alone (25mg/kg, i.p., qd x 5) resulted in only about 20% inhibition of tumour growth (Table 3). Thus, liposomal DNA/5-FU prodrug combinations resulted in s.c. tumour regressions.
  • mice and mice treated with 5-FU (30 mg/kg i.p., qd ⁇ 5) died from tumour by 30 days.
  • CMV-BL/1614W94 treatment increased survival to 60%
  • CMV-CD/5-FC treatment also increased the survival to 40% (Table 4).

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Abstract

L'invention concerne une chimère moléculaire conçue pour être utilisée en thérapie avec un promédicament et comprenant une séquence d'ADN de régulation de transcription capable d'être activée dans une cellule mammifère ciblée, ainsi qu'une séquence de codage d'ADN reliée à ladite séquence d'ADN de régulation de transcription et codant pour une β-lactamase, de sorte que, lorsque ladite séquence de codage est exprimée dans la cellule ciblée, ladite enzyme est capable de catalyser la conversion du promédicament en un agent toxique pour la cellule ciblée. Cette β-lactamase est particulièrement avantageuse, quand on l'utilise dans GDEPT ou VDEPT dans la gamme de médicaments pouvant être produits et qui sont capables d'être convertis en espèces actives par l'enzyme.
PCT/GB1996/002845 1995-11-20 1996-11-19 Vecteur constitue d'une sequence d'adn de regulation de transcription liee a une sequence d'adn codant pour une beta-lactamase pour therapie a base d'un promedicament enzymatique WO1997019180A2 (fr)

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AU75839/96A AU7583996A (en) 1995-11-20 1996-11-19 Vector consisting of a transcriptional regulatory dna sequence linked to a dna sequence encoding beta-lactamase for enzyme prodrug therapy

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PCT/GB1996/002845 WO1997019180A2 (fr) 1995-11-20 1996-11-19 Vecteur constitue d'une sequence d'adn de regulation de transcription liee a une sequence d'adn codant pour une beta-lactamase pour therapie a base d'un promedicament enzymatique

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AU7583996A (en) 1997-06-11
WO1997019180A3 (fr) 1997-08-28

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